Xav939 wnt inhibitor

E14 WT ESC grown in serum + LIF were treated with 2 mL TrypLE Express (Life Technologies). Cells were centrifuged (160 rcf) and then resuspended in 5 mL of N2B27 with 0.1% of BSA (Life Technologies) to get a single cell suspension. Next, cells were counted with the BioRad TC20 cell counter and 15,000 cells/cm² were plated in 10 mL of N2B27 with 0.1% of BSA and 10 ng/mL of bFgf (PeproTech, 100-18B). Cells were differentiated for five days and media was changed every day without any PBS washings in between: Day1 (10 mL of N2B27 with 0.004% of BSA and 10 ng/mL bFgf).; Day2 (10 mL of N2B27 with 0.004% of BSA, 10 ng/mL bFgf and 5 μM Xav939/Wnt inhibitor (Sigma-Aldrich, x3004-5mg)); Day3 (10 mL of N2B27 with 0.004% of BSA and 5 μM Xav939/Wnt inhibitor); Day4 (10 mL of N2B27 with 0.004% of BSA and 5 μM Xav939/Wnt inhibitor). On Day 5, cells were washed 1–2 with PBS, and collected for downstream analyses. Differentiation was assessed using RT-qPCR on a Light Cycler 480II comparing AntNPC d5 vs. E14 WT d0 relative gene expression levels (using the 2ΔCt method) with housekeeping gene (Eef1a1), pluripotency markers (Pou5f1/Oct4 and Nanog), mesoderm marker (T) and ectoderm markers (Six3 and Lhx5). Standard deviations were calculated from technical triplicate reactions and were represented as error bars.